This invention relates to a method for the quantitative determination or assay of glutamate oxalacetate transaminase or glutamate pyruvate transaminase present in a biological fluid, especially in human blood serum. More particularly, the invention relates to a method in which L-glutamate produced by transamination in the course of the determination is oxidatively deaminated in an oxidation-reduction reaction with simultaneous production of reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the content of the transaminase in the fluid, and to an improvement for obviating the interference caused by another substance in the fluid which while not related quantitatively to the substance being determined undergoes an oxidation-reduction reaction with the production of reduced beta-nicotinamide adenine dinucleotide and thus reduces the accuracy of the determination. The invention also relates to a reagent combination for use in the method.
The activities of the enzymes glutamate oxalacetate transaminase (GOT), also known as aspartate aminotransferase, and glutamate pyruvate transaminase (GPT), also known as alanine aminotransferase, in biological fluids in the past have been determined by the indirect measurement of one of the products of a transamination reaction catalyzed by the transaminase to be determined. A method having certain advantages embodies the production of L-glutamate from alpha-ketoglutarate (2-oxoglutarate) by transamination. The alpha-ketoglutarate is reacted with L-aspartate in the determination of GOT, and with L-alanine in the determination of GPT. In an oxidation-reduction reaction of the L-glutamate with beta-nicotinamide adenine dinucleotide in its oxidized form (NAD), conducted in the presence of the enzyme glutamate dehydrogenase (GLDH) as catalyst, the L-glutamate is oxidatively deaminated, and the NAD is converted to the reduced form (NADH) of the compound. The quantity of NADH produced is proportional to the activity of the transaminase in the biological fluid. The NADH is reacted with 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT) in an oxidation-reduction reaction, to produce the reduced form (INTH) of the latter compound, proportionally to the activity of the transaminase in the fluid. The INTH may be determined quantitatively by measurement of light absorption, preferably at the absorption maximum of 500 nanometers (nm.). The color is very stable and intense, therefore sensitive, and the absorbance is proportional to the concentration of GOT or GPT in the fluid over a large dynamic range.
The foregoing reactions, which have been performed as coupled reactions by incubation of the biological fluid with a single mixed reagent, may be represented as follows: EQU Alpha-ketoglutarate+L-aspartate.sup.GOT L-glutamate+oxalacetate (1A.)
or EQU Alpha-ketoglutarate+L-alanine.sup.GPT L-glutamate+pyruvate (1B.) EQU L-Glutamate+NAD+H.sub.2 O.sup.GLDH alpha-ketoglutarate+NADH+NH.sub.3 ( 2.) EQU NADH+INT.fwdarw.NAD+INTH (3.)
where:
GOT=Glutamate oxalacetate transaminase PA1 GPT=Glutamate pyruvate transaminase PA1 GLDH=Glutamate dehydrogenase PA1 NAD=Beta-nicotinamide adenine dinucleotide PA1 NADH=Reduced form of NAD PA1 INT=2-p-Iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride PA1 INTH=Reduced form of INT
Reaction 3 may be catalyzed non-enzymatically, by N-methyl phenazonium methosulfate (PMS--also called phenazine methosulfate), or enzymatically, by diaphorase. The use of diaphorase is preferred, as diaphorase catalyzes the reaction more rapidly, is not affected by light, and is not auto-oxidizable as is PMS. The use of diaphorase also is adaptable to making kinetic measurements of the transaminase activity by following the rate of INTH color formation with time.
While the foregoing method is advantageous, the biological fluids contain endogenous materials which participate in enzymatic oxidation-reduction reactions with the formation of NADH, thereby interfering with the determination. The equilibrium of reaction 2 lies toward the formation of L-glutamate at the reaction pH of about 7-8. Consequently, large concentrations of GLDH are required in order to obtain results which are linear in respect to the concentration of GOT or GPT present. In the presence of large concentrations of GLDH, certain amino acids in the fluids undergo oxidative reactions which result in equivalent production of NADH, owing to the low specificity of the enzyme GLDH. The non-specific NADH produced from such amino acids results in non-specific color due to resulting INTH production and a false elevation of the transaminase activity in the biological fluid. Owing to the high degree of variability in amino acid content of different fluid samples, the false elevation is unpredictable and renders many of the determinations by this procedure valueless.
Also present in biological fluids, particularly serum, are lactate and the enzyme lactate dehydrogenase (LDH), in variable concentrations. Owing to the activity of the enzyme, these substances increase the content of NADH in the assay medium. In the past, oxamate or oxalate has been added to the medium, to inhibit lactate dehydrogenase.
Several other procedures are available to increase the reliability of results obtained with biological fluids containing interfering endogenous substances. Thus, a blank determination can be run for each sample of the fluid, using a reagent which omits the alpha-ketoglutarate. This procedure substantially increases the cost in labor and reagent material. The determination may be preceded by removing L-glutamate from the fluid, with the enzyme glutamate decarboxylase. However, this enzyme does not remove other substances which also are substrates for GLDH. Another mehod which may, to a point, correct for the non-specific color resulting from the oxidation of endogenous substances would be to take readings at two different times, rendering the determination a kinetic one. This procedure would not lend itself conveniently to performance of the determination in automated instruments.